Recombinant expression and functional characterization of Aspergillus niger β-glucosidase in Pichia pastoris for enhanced synergistic saccharification of lignocellulosic biomass toward biofuel applications

Efficient conversion of lignocellulosic biomass into fermentable sugars is crucial for sustainable biofuel production and the utilization of agricultural residues. In this study, the β-glucosidase gene ( bg1 ) from Aspergillus niger was heterologously expressed in Pichia pastoris X-33 under the AOX1 promoter to assess its potential for biomass saccharification. Successful cloning and genomic integration of the bg1 gene were confirmed through restriction digestion and PCR analysis. Enzyme expression and secretion were verified by SDS-PAGE and esculin agar assay, which revealed a prominent protein band at approximately 60 kDa, indicating successful production of recombinant Bg1. Optimization of methanol induction showed that 1% methanol supported maximum enzyme production, with the highest β-glucosidase activity on day 6. The recombinant enzyme exhibited optimal activity at pH 5.5 and 70 °C and showed high thermal stability, retaining over 96% residual activity after 72 h at 60-70 °C. Enzymatic saccharification using recombinant β-glucosidase combined with recombinant endoglucanase resulted in high saccharification efficiencies of 96%, 86%, and 94% for sugarcane bagasse, wheat straw, and rice straw, respectively, after 72 h. Structural and physicochemical analyses using SEM, FT-IR, and XRD confirmed significant morphological disruption, chemical modifications, and increased crystallinity of the treated biomass. Overall, the findings demonstrate the functional expression and biochemical properties of recombinant Bg1 and its contribution, in combination with recombinant endoglucanase, to the saccharification of physically processed agricultural residues under laboratory-scale conditions.